We have continued our research on membrane fusion, the fundamental step in secretion, viral infection, fertilization and neuro-transmission. Sea urchin egg cortical granule exocytosis, an example of calcium triggered membrane fusion, has served as a model system. We have been investigating how certain proteins (NSF et al.) might regulate the exocytosis of the egg's cortical granules. We have established that cytosol is unable to restore fusion to the isolated exocytotic machinery inactivated by NEM. We have found using immunoblot analysis that sea urchin eggs contain NSF in their cytosol but that this protein is absent from an in vitro preparation which undergoes fusion upon the provision of Ca2+. However, a stable and specific interaction between NSF and the isolated exocytotic machinery can occur under the appropriate conditions. These results suggest that the role of NSF might be in the establishment of a fusion-competent arrangement rather than in membrane fusion per se: a novel, NSF-independent mechanism of fusion. The relative roles of inactivation and heterogeneity were evaluated further in the isolated planar cortex from the sea urchin eggs using multiple solution exchange protocols and perfusion at different rates. No evidence was found to support the hypothesis that either rate-dependent or rate-independent inactivation is responsible for the cessation of fusion at calcium concentrations which produced sub-maximal responses in cortical degranulation. Calcium-triggered exocytosis was heterogeneous: exocytotic responses were sub-maximal at certain calcium concentrations because all the granules capable of fusing at those concentrations of calcium had fused. Secretory models featuring calcium-dependent inactivation are inappropriate for describing cortical degranulation in the sea urchin egg. The minimum number of calcium ions involved in the fusion process was estimated by considering the behavior of a single, step-like transition at a threshold calcium concentration. This analysis suggests that more than four calcium ions are involved in triggering exocytosis. Our model, which describes both the kinetic and steady-state features observed in sea urchin cortical granule exocytosis, has been extended with the addition of a third parameter representing the activation time for fusion complexes. Discrepancies between the model and the initial time course of exocytosis have been eliminated with this modification.